Method for the isolation and presumptive diagnosis of Bacillus cereus and tellurite medium therefor

ABSTRACT

A method is provided for the isolation and presumptive diagnosis of Bacillus cereus which method is useful in the assay of antibiotic preparations, food products such as milk, drug products and cosmetics. A medium for use in such method is also provided which medium includes tellurite, glycine and polymyxin, and inhibits most organisms which grow and resemble Bacillus cereus on this medium. If an organism grows on the medium and has characteristic colonies, a presumptive diagnosis of Bacillus cereus can be assumed until further tests are made.

FIELD OF THE INVENTION

The present invention relates to a method for the isolation and presumptive diagnosis of Bacillus cereus and to a medium for use in such method.

BACKGROUND OF THE INVENTION

Strains of Bacillus cereus have been implicated in cases of food poisoning in humans and in bovine mastitis coming, in some instances, from antibiotics used in treatment. Thus, a medium for the rapid presumptive diagnosis of Bacillus cereus would indeed be useful. In fact, several such media have been described including egg yolk, citrate, lithium chloride and polymyxin, as described by Donovan, K. O., "A Selective Medium for Bacillus cereus in Milk", J. Appl. Bacteriol., 21:100-103 (1958); mannitol, egg yolk and polymyxin, as described by Kim et al., "Occurrence of Bacillus cereus in Selected Dry Food Products", J. Milk Food Technol. 34:12-15 (1971); egg yolk and polymyxin as described by Kim et al., "Enumeration and Identification of Bacillus cereus in Foods", Appl. Microbiol. 22:581-587 (1971); and mannitol, egg yolk and phenol red agar as described by Mossel, et al., "Enumeration of Bacillus cereus in Foods," Appl. Microbiol., 15:650-653 (1967).

The above media are all egg yolk containing media. Unfortunately, unless such media are handled with extreme care, a troublesome spreading problem may result.

The use of tellurite as an inhibitor was reported by Fleming, A. F., "On the Specific Antibacterial Properties of Penicillin and Potassium Tellurite", J. Pathogenic. Bacteriol., 35:831-842 (1932). Zebovitz et al. report the use of tellurite (and 3% glycerine) for coagulase positive staphylococci, in "Tellurite-Glycine Agar:A Selective Plating Medium for the Quantitative Detection of Coagulase-Positive Staphylococci", J. Bacteriol, 70:686-690 (1955).

DESCRIPTION OF THE INVENTION

As indicated, Bacillus cereus has been found to be responsible for outbreaks of food poisoning in many parts of the world. It would thus be of enormous value to be able to follow the proliferation of Bacillus cereus in foods, drugs, cosmetics and the like so as to establish, before any disease may occur, that the food or other comestible or cosmetic may become dangerous upon further storage.

In accordance with the present invention, a method is provided for the isolation and presumptive diagnosis of Bacillus cereus in milk, antibiotic preparations, other food and drug products or cosmetics, which method includes the steps of preparing a non-egg yolk-containing medium for growth of Bacillus cereus which inhibits most organisms which grow and resemble Bacillus cereus on such medium, the medium including tellurite, glycine and polymyxin, and assaying a product such as described above employing said medium to isolate Bacillus cereus, if present in said assayed product, on said medium, and allowing for presumptive diagnosis of Bacillus cereus. Thus, if an organism grows on the medium and has characteristic colonies, a presumptive diagnosis of Bacillus cereus can be assumed until further tests are made. The method of the invention is effective in differentiating Bacillus cereus from similarly appearing organisms. However, the final diagnostic criteria must be made only after other differential tests have been completed. Notwithstanding the above, it is still extremely useful to have a rapid presumptive procedure as described herein which employs a medium which does not contain egg yolk and therefore does not promote spreading.

In addition, in accordance with the present invention, an agar (non-egg-yolk containing) medium is provided which includes in addition to agar and other conventional nutrients, tellurite preferably in the form of potassium tellurite, glycine and polymyxin, and optionally mannitol.

The tellurite will be present in an amount within the range of from about 0.005 to about 0.02% and preferably from about 0.01 to about 0.015% by weight of the medium. The presence of more than about 0.01% by weight tellurite is unnecessary and will have no bearing on organism inhibition while the presence of less than about 0.005% by weight tellurite will allow for growth of black colonies which are characteristic of but different from Bacillus cereus colonies.

The glycine will be present in the medium in a weight ratio to the tellurite of within the range of from about 2500:1 to about 3000:1 and preferably from about 2900:1 to about 3000:1 and in an amount within the range of from about 2 to about 4% and preferably from about 2.5 to about 3.5% by weight of the medium. Where less than about 1.5% glycine is present, certain staphylococci will grow on tellurite-containing media with the colonies somewhat resembling Bacillus cereus (small, black). However, the presence of more than 3% by glycine is unnecessary and will not add to the efficacy of the invention.

Polymyxin will be present in the medium of the invention in a weight ratio to the tellurite of within the range of from about 0.05:1 to about 0.2:1 and preferably from about 0.09:1 to about 0.1:1 and in an amount within the range of from about 0.0005 to about 0.0015% and preferably from about 0.0009 to about 0.001% by weight. Where less than about 0.0001% by weight polymyxin is present, the medium could support the growth of other organisms. However, the presence of more than about 0.001% by weight polymyxin is unnecessary and produces no significant advantages.

Mannitol fermentation (acid) may be included in the medium of the invention in amounts within the range of from about 0.4 to about 0.6% and preferably from about 0.49 to about 0.51% by weight. The mannitol will indicate growth of organisms which may ferment mannitol.

The medium of the invention will also contain from about 1 to about 2% by weight agar and/or other conventional nutrients for supporting the growth of Bacillus cereus. Examples of such materials which may be present in the agar include, but are not limited to, surfactants such as Tween 20, as well as phenol red, serum, and the like.

In carrying out the method of the invention, the tellurite-glycine-polymyxin agar medium is prepared. Thereafter, the material to be assayed is grown on the medium employing the following procedure.

Inoculate enrichment medium with 1.0 to 10.0 ml of an appropriate sample (food, drug or other sample). Incubate 24 hours at 37° C., and observe for growth. Inoculate tellurite-glycine-polymyxin agar plates with 0.1 ml of enrichment medium, spread the inoculum, and again incubate at 37° C. until growth is observed. Inoculation can be made to plates direct from samples, if desired, omitting the enrichment.

The agar medium is then observed for the presence of colonies and colonial characteristics, particularly black coloration of colonies which indicate Bacillus cereus.

EXAMPLE

The following experiments were carried out to illustrate the method for the isolation and presumptive diagnosis of Bacillus cereus.

    ______________________________________                                         Materials                                                                      ______________________________________                                         1.   Organisms used                                                                 All organisms bearing S.C. designation (Squibb                                 culture) have been thoroughly classified in                                    Squibb Laboratories. Others are well known                                     strains. -2. Media:                                                       A.     Enrichment Medium                                                              Brain Heart Infusion (BBL)                                                                           37       g                                               Glycine               30       g                                               Tween 20              100      g                                               Water                 + 0 1000 ml                                       Sterilize at 121.5° C. for 15 miniutes, (cool                           to 50° C.) and add 1 ml of a 10,000 mcg/ml                              solution of filter sterilized polymyxin                                        B sulfate. Dispense 50 ml to sterile 250 ml                                    cotton stoppered Erlenmeyer flasks.                                            B.     Tellurite-Glycine-Polymyxin Agar                                               Trypticase Soy Broth (BBL-No                                                                         27.5     g                                               dextrose)                                                                      Agar Granulated (BBL) 15.0     g                                               Mannitol              5.0      g                                               Glycine               30.0     g                                               Phenol Red            0.018    g                                               Water                 to 940.0 ml                                              Mix and sterilize at 121.5° C. for 15                                   minutes; cool to 50° C. and add:                                        Sterile 10,000 mcg/ml polymyxin                                                                      1.0      ml                                              B sulfate                                                                      Sterile Human Serum   50.0     ml                                              Sterile 1% Potassium  10.0     ml                                              Tellurite Solution                                                             Pour 20 ml into 100 mm Petri plates.                                    C.     Brain Heart Infusion Broth                                                     Brain Heart Infusion  37.0     g                                               Distilled water       to 100.0 ml                                              Dissolve and dispense 20 ml to 1 × 6"                                    screw cap tubes. Sterilize at 121.5° C.                                 for 15 minutes. -D.   Tellurite Agar without Glycine                           Medium B without glycine                                                E.     Saline                                                                         0.9% NaCl in water.                                                     3.   Filter System                                                                  Use a Nalgene No. 120, 0.2 M                                              ______________________________________                                    

Methods

1. Samples

A. Antibiotic Powder--use 1 g

B. Cloxacillin Syringes--use contents (6 ml)

C. Fungizone Oral Suspension--use 10 ml of reconstituted powder

D. Cultures--1 ml of BHI grown cells to Medium A or 0.5 ml to Medium B

2. Culture Methods

A. Stock Culture Broth

Inoculate Medium C with a loop of stock culture and incubate at 37° C. for 24 hours.

B. Enrichment Culture

Inoculate Medium A with sample as above.

Incubate 24 hours at 37° C. on a flask shaker.

Where desired, 1.0 ml of Penicillinase

Solution (BBL-10⁶ μ/ml) can be added for cloxacillin syringe culture.

C. Tellurite Agar Culture

Inoculate plates with 0.5 ml of Enrichment Broth or Stock culture broth, spread with a glass rod, and incubate at 37° C.

3. Test readings

A. Broth

Observe flasks or tubes for turbidity.

B. Agar

Observe plates for the presence of colonies and colonial characteristics, particularly black coloration of colonies. Observe areas around colonies for clear zones in the red agar, indicating mannitol fermentation.

4. Sensitivity of the Method

Serially dilute B. cereus ATCC 11778, in Medium C to 10⁹, in sterile physiological saline (Medium E). Transfer 1.0 ml of each dilution to tubes containing 10 ml molten BHI Agar, cooled to 50° C. Mix and pour into 100 mm Petri plates. Incubate overnight and count colonies. Transfer 1.0 ml from each dilution to the contents of a cloxacillin syringe contained in separate sterile blender flasks (100 ml). Blend 30 seconds and add 50 ml Medium A. Again blend for 30 seconds. Transfer each of the contents to separate sterile 250 ml Erlenmeyer flasks and incubate overnight at 37° C. Observe for growth and transfer 0.5 ml of each flask to separate plates of Medium B. Again, incubate at 37° C., but observe plates for growth for up to 72 hours.

Results

As shown in Table One, many organisms similar to B. cereus did not grow in the presence of polymyxin (Medium A). In addition, the presence of tellurite was inhibitory to still other organisms. Of these organisms which can grow on Media A and B, most are readily differentiated from B. cereus by colonial appearance. The remaining few are further differentiated by mannitol fermentation (acid).

Certain of the staphylococci grow on tellurite-containing media with the colonies somewhat resembling B. cereus (small, black). To separate these strains from B. cereus, glycine was added at 3%. Table Two shows growth on Medium B with and without glycine, compared to growth of B. cereus. Should growth of other strains be found, mannitol fermentation would again be a useful differential test.

Table Three shows the potential usefulness of tellurite-glycine medium in isolation of B. cereus from antibiotic preparations with and without the addition of B. cereus. In addition, milk can also be evaluated for the presence of B. cereus by the same method.

Sensitivity of the method, comparing plate counts of serial dilutions on Brain Heart Infusion Agar to antibiotic samples to which serially diluted B. cereus had been added, indicates a sensitivity of less than five cells/sample.

In order to determine typical B. cereus cells on tellurite-glycine-polymyxin Medium B, B. cereus cells were prepared by extrusion of a sample, in Medium A, of spiked cloxacillin syringes through an 0.2/μ filter (syringe type). Black colonies indicate B. cereus, while the gray colonies, by microscopy, are fungi. Since these syringes (see method 4), were spiked at a level of five cells/syringe, the sensitivity of the method is confirmed.

                                      TABLE ONE                                    __________________________________________________________________________     GROWTH AND APPEARANCE OF MICROORGANISMS ON TELLURITE MEDIA                                                                        Medium                      Test                           Growth      Appearance                                                                             Acid From                   No.                                                                               Organism   ATCC Number                                                                            Squibb Number                                                                           Medium A                                                                             Medium B                                                                             On Medium B                                                                            Mannitol                    __________________________________________________________________________     1  Bacillus cereus                                                                           13061   8598     +     +     SB      None                        2  B. cereus  11778   8613     +     +     SB      None                        3  B. circulans                                                                              --      1834     NG    NG    NG      --                          4  B. coagulans                                                                              7050    9261     NG    NG    NG      --                          5  B. megaterium                                                                             14946   3782     +     +     LG      +                           6  B. megaterium                                                                             10778   3783     NG    NG    NG      --                          7  B. polymyxa                                                                               NRRL-B-510                                                                             1522     +     +     SB      +                           8  B. pumilis 14884   8513     NG    NG    NG      --                          9  B. subtilis                                                                               6633    1355     NG    NG    NG      --                          10 B. subtilis                                                                               NRRL-558                                                                               1659     NG    NG    NG      --                          11 B. subtilis                                                                               12139   3882     +     +     SB      +                           12 B. subtilis                                                                               6455    3409     + (trace)                                                                            NG    NG      --                             (var. niger)                                                                13 B. thuringensis                                                                           10792   2928     NG    NG    NG      --                          14 Corynebacterium                                                                           NCTC 9755                                                                              3638     +     +     LG      ND                             xerosis                                                                     15 C. acnes   6919    4020     +     +     LG      ND                          16 Brevibacterium                                                                            6872    9149     +     +     Brown   ND                             ammoniagena                                                                 17 B. cereus  --      local isolant                                                                           +     +     SB      None                        18 B. cereus  --      Philpot Number 1                                                                        +     +     SB      None                                              (local isolant)                                          19 Saliva Control     --       +     one   White (yeast)                                                                          None                           (for Streptococcus sp)            colony                                    __________________________________________________________________________      Code: SB = small, black colonies (1-3 mm); LG = large, gray colonies (>5       mm); NG = No growth; ND = not determined                                 

                                      TABLE TWO                                    __________________________________________________________________________     GROWTH APPEARANCE OF STAPHYLOCOCCUS AUREUS AND                                 B. CEREUS ON TELLURITE MEDIA                                                                             Growth on Medium B                                   Organism   ATCC Number                                                                            SC Number                                                                             Glycine                                                                            No Glycine                                                                           BHI Agar (BBL)                             __________________________________________________________________________     Staphylococcus aureus                                                                     --      2399   0   +     +                                          S. aureus  --      1579   0   +     +                                          S. aureus  --      9578   0   +     +                                          S. aureus  --      9601   0   +     +                                          S. aureus  --      local isolant                                                                         0   +     +                                          B. cereus  10361   8598   +   +     +                                          B. cereus  11778   8613   +   +     +                                          B. cereus  --      Philpot #1                                                                            +   +     +                                          __________________________________________________________________________

                  TABLE THREE                                                      ______________________________________                                         CONTAMINATION OF PRODUCTS BY B. CEREUS                                         Sample              Growth                                                     Number     Contents Medium A      Medium B                                     ______________________________________                                         1          FOS      NG            NG                                           2          FOS      NG            NG                                           3          FOS      NG            NG                                           4          FOS      NG            NG                                           5          FOS.sup.a                                                                               +             SB                                           6          FOS.sup.a                                                                               +             SB                                           7          Ampho    NG            NG                                           8          Ampho.sup.a                                                                             +             SB                                           9          Clox     NG            NG                                           10         Clox     NG            NG                                           11         Clox     NG            NG                                           12         Clox     NG            NG                                           13         Clox.sup.a                                                                              +             SB                                           14         Clox.sup.a                                                                              +             SB                                           15         Clox.sup.a                                                                              +             SB                                           16         Clox.sup.a                                                                              +             SB                                           17         Milk.sup.a                                                                              +             SB                                           18         Milk.sup.a                                                                              +             SB                                           ______________________________________                                          CODE:                                                                          FOS = E. R. Squibb & Sons, Inc. Fungizone for Oral Suspension  Batch           Number 174KL                                                                   Ampho = E. R. Squibb & Sons, Inc. Amphotericin Powder  FGNX17-CP               Clox = 250 mg cloxacillin in 6 ml peanut oil, E. R. Squibb & Sons, Inc.        Cloxacillin Syringe for Udder Infusion                                         .sup.a = spiked with approximately five cells of B. cereus ATCC 11778 per      dose 1 g or one syringe (6 ml)                                                 SB = small, black colonies                                                

What is claimed is:
 1. A method for the isolation and presumptive diagnosis of Bacillus cereus which comprises preparing an agar medium for growth of Bacillus cereus which medium is free of egg yolk and comprises a tellurite salt, glycine and polymyxin, the tellurite salt, glycine and polymyxin being present in amounts which inhibit most organisms which grow and resemble Bacillus cereus on such medium, growing a sample on said medium employing said medium to isolate Bacillus cereus, determining the presence of Bacillus cereus whereby a presumptive diagnosis of Bacillus cereus can be made if an organism grows on the medium.
 2. The method as defined in claim 1 wherein the tellurite salt is potassium tellurite.
 3. The method as defined in claim 1 wherein the tellurite salt is present in an amount within the range of from about 0.01 to about 0.015% by weight of the medium.
 4. The method as defined in claim 1 wherein the glycine is present in an amount within the range of from about 2 to about 4% by weight of the medium.
 5. The method as defined in claim 1 wherein the polymyxin is present in an amount within the range of from about 0.0005 to about 0.0015% by weight of the medium.
 6. The method as defined in claim 1 wherein the medium further includes mannitol, phenol red, serum, trypticase soy, and agar.
 7. The method as defined in claim 1 wherein the glycine is present in a weight ratio to the tellurite of within the range of from about 2500:1 to about 300:1 and the polymyxin is present in a weight ratio to the tellurite of within the range of from about 0.09:1 to about 0.1:1.
 8. The method as defined in claim 1 wherein said sample to be grown on said medium is a food product, drug product or cosmetic.
 9. The method as defined in claim 8 wherein said sample to be grown on said medium is an antibiotic preparation or milk. 